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BACKGROUND AND AIM Epigenetic modification is an important part of the pathogenesis of inflammatory bowel disease (IBD). Some studies proved that p62 was involved in inflammatory response and upregulated in IBD patients, and histone modification plays an important role in regulating p62 expression. SETD8, a histone H4K20 methyltransferase, has been reported downregulated in some inflammatory diseases. Here, we investigated the role of SETD8 in the development of IBD and its underlying mechanisms. METHODS An inflammatory cell model was established to elucidate whether SETD8 involved in inflammatory response in macrophages. Three percent dextran sodium sulfate-induced colitis murine model injection with SETD8 inhibitor was used in our study to investigate whether SETD8 inhibition can affect the progress of IBD. The expression of SETD8 and p62 was measured by qRT-PCR and western blot. The mRNA level of inflammatory cytokines was analyzed by qRT-PCR. In addition, chromatin immunoprecipitation-PCR was performed to identify the mechanism by which SETD8 regulates p62. RESULTS SETD8 expression obviously decreased in vitro, in vivo models and in IBD patients. In lipopolysaccharide-activated RAW264.7 cells, knockdown of SETD8 significantly increased the mRNA expression of inducible nitric oxide synthase, cyclooxygenase-2, TNF-α, IL-6, IL-1β, and MCP-1. Based on the dataset, we verified that p62 was a target gene of SETD8 and chromatin immunoprecipitation-PCR assay identified that silence of SETD8 distinctly decreases the H4K20me1 enrichment in the promoter of p62. Moreover, silencing of p62 partly reverses the SETD8 inhibition-mediated pro-inflammatory effect in vitro. Finally, SETD8 pharmacological inhibitor (UNC0379) aggravated the disease progression in dextran sodium sulfate-induced murine colitis. CONCLUSION Our findings elucidate an epigenetic mechanism by which SETD8 regulates the p62 expression and restrains the inflammatory response in colitis. Our result suggests that targeting SETD8 may be a promising therapy for IBD.

MLL3 is a histone H3K4 methyltransferase that is frequently mutated in cancer, but the underlying molecular mechanisms remain elusive. Here, we found that MLL3 depletion by CRISPR/sgRNA significantly enhanced cell migration, but did not elevate the proliferation rate of cancer cells. Through RNA-Seq and ChIP-Seq approaches, we identified TNS3 as the potential target gene for MLL3. MLL3 depletion caused downregulation of H3K4me1 and H3K27ac on an enhancer ~ 7 kb ahead of TNS3. 3C assay indicated the identified enhancer interacts with TNS3 promoter and repression of enhancer activity by dCas9-KRAB system impaired TNS3 expression. Exogenous expression of TNS3 in MLL3 deficient cells completely blocked the enhanced cell migration phenotype. Taken together, our study revealed a novel mechanism for MLL3 in suppressing cancer, which may provide novel targets for diagnosis or drug development.

Gene transcription is coordinately regulated by multiple transcription factors. However, a systematic approach is still lacking to identify co-regulators for transcription factors. Here, we performed ChIP-Seq analysis and predicted the regulators for p53-mediated transcription process, from which we confirmed the roles of GLIS2, MAZ and MEF2A in regulating p53 target genes. We revealed that GLIS2 selectively regulates the transcription of PUMA but not p21. GLIS2 deficiency caused the elevation of H3K27ac and p53 binding on the PUMA enhancer, and promoted PUMA expression. It increased the rate of apoptosis, but not cell cycle. Moreover, GLIS2 represses H3K27ac level on enhancers, regulates the gene expression related with focal adhesion and promotes cell migration, through inhibiting p300. Big data analysis supports GLIS2 as an oncogene in colon cancer, and perhaps other cancers. Taken together, we have predicted candidates for p53 transcriptional regulators, and provided evidence for GLIS2 as an oncogene through repressing enhancer activation.

Enhancer is one kind of cis-elements regulating gene transcription, whose activity is tightly controlled by epigenetic enzymes and histone modifications. Active enhancers are classified into typical enhancers, super-enhancers and over-active enhancers, according to the enrichment and location of histone modifications. Epigenetic factors control the level of histone modifications on enhancers to determine their activity, such as histone methyltransferases and acetylases. Transcription factors, cofactors and mediators co-operate together and are required for enhancer functions. In turn, abnormalities in these trans-acting factors affect enhancer activity. Recent studies have revealed enhancer dysregulation as one of the important features for cancer. Variations in enhancer regions and mutations of enhancer regulatory genes are frequently observed in cancer cells, and altering the activity of onco-enhancers is able to repress oncogene expression, and suppress tumorigenesis and metastasis. Here we summarize the recent discoveries about enhancer regulation in cancer and discuss their potential application in diagnosis and treatment.

In eukaryote cells, core components of chromatin, such as histones and DNA, are packaged in nucleus. Leakage of nuclear materials into cytosol will induce pathological effects. However, the underlying mechanisms remain elusive. Here, cytoplasmic localization of nuclear materials induced by chromatin dysregulation (CLIC) in mammalian cells is reported. H3K9me3 inhibition by small chemicals, HP1α knockdown, or knockout of H3K9 methylase SETDB1, induces formation of cytoplasmic puncta containing histones H3.1, H4 and cytosolic DNA, which in turn activates inflammatory genes and autophagic degradation. Autophagy deficiency rescues H3 degradation, and enhances the activation of inflammatory genes. MRE11, a subunit of MRN complex, enters cytoplasm after heterochromatin dysregulation. Deficiency of MRE11 or NBS1, but not RAD50, inhibits CLIC puncta in cytosol. MRE11 depletion represses tumor growth enhanced by HP1α deficiency, suggesting a connection between CLIC and tumorigenesis. This study reveals a novel pathway that heterochromatin dysregulation induces translocation of nuclear materials into cytoplasm, which is important for inflammatory diseases and cancer., The current study reveals a novel pathway leading to cytoplasmic localization of nuclear material induced by chromatin dysregulation (CLIC). HP1α knockdown, SETDB1 knockout or H3K9me3 inhibition by drugs, induces cytoplasmic puncta containing histones H3.1, H4 and DNA, which is regulated by MRE11 and NBS1. CLIC activates inflammatory gene expression, γH2AX formation, and autophagy, and is related with inflammation and tumorigenesis.

Background The aetiology of inflammatory bowel disease (IBD) is related to genetics and epigenetics. Epigenetic regulation of the pathogenesis of IBD has not been well defined. Here, we investigated the role of H3K27ac events in the pathogenesis of IBD. Based on previous ChIP-seq and RNA-seq assays, we studied signal transducer and activator of transcription 1 (STAT1) as a transcription factor (TF) and investigated whether the STAT1–EP300–H3K27ac axis contributes to the development of IBD. We performed ChIP-PCR to investigate the interaction between STAT1 and H3K27ac, and co-IP assays were performed to investigate the crosstalk between STAT1 and EP300. Results Lymphocyte cytosolic protein 2 (LCP2) and TNF-α‐inducible protein 2 (TNFAIP2) are target genes of STAT1. p-STAT1 binds to the enhancer loci of the two genes where H3K27ac is enriched, and EP300 subsequently binds to regulate their expression. In mice with dextran sulfate sodium (DSS)-induced acute colitis, an EP300 inhibitor significantly inhibited colitis. Conclusions p-STAT1 and EP300 promote TNFAIP2 and LCP2 expression through an increase in H3K27ac enrichment on their enhancers and contribute to the pathogenesis of chronic inflammation. Graphic abstract

Colorectal cancer (CRC) is one of the most common cancers in the world. Although genomic mutations and SNPs have been extensively studied, the epigenomic status in CRC patient tissues remains elusive. Here, we profiled active enhancers genome-widely in paired CRC patient tissues through H3K27ac ChIP-Seq, together with genomic and transcriptomic analysis. Totally we sequenced 73 pairs of CRC tissues and generated 147 H3K27ac ChIP-Seq, 144 RNA-Seq, 147 whole genome sequencing and 86 H3K4me3 ChIP-Seq files. Our analysis discovered 5590 gain variant enhancer loci (VEL) and 1100 lost VELs in CRC, and 334 gain variant super enhancer loci (VSEL) and 121 lost VSELs. Multiple key transcription factors in CRC were predicted with motif analysis and core regulatory circuitry analysis. Further experiments verified the functions of 6 super enhancers governing PHF19, LIF, SLC7A5, CYP2S1, RNF43 and TBC1D16 in regulating cancer cell migration, and we identified KLF3 as a novel oncogenic transcription factor in CRC. Taken together, our work provides important epigenomic resource and novel functional factors for epigenetic studies in CRC.

Background and aims Trimethylation of Lys36 on histone 3 (H3K36me3) catalyzed by histone methyltransferase SET domain-containing 2 (SETD2) is one of the most conserved epigenetic marks from yeast to mammals. SETD2 is frequently mutated in multiple cancers and acts as a tumor suppressor. Approach and results Here, using a liver-specific Setd2 depletion model, we found that Setd2 deficiency is sufficient to trigger spontaneous HCC. Meanwhile, Setd2 depletion significantly increased tumor and tumor size of a diethylnitrosamine-induced HCC model. The mechanistic study showed that Setd2 suppresses HCC not only through modulating DNA damage response, but also by regulating lipid metabolism in the liver. Setd2 deficiency down-regulated H3K36me3 enrichment and expression of cholesterol efflux genes and caused lipid accumulation. High-fat diet enhanced lipid accumulation and promoted the development of HCC in Setd2-deficient mice. Chromatin immunoprecipitation sequencing analysis further revealed that Setd2 depletion induced c-Jun/activator protein 1 (AP-1) activation in the liver, which was trigged by accumulated lipid. c-Jun acts as an oncogene in HCC and functions through inhibiting p53 in Setd2-deficient cells. Conclusions We revealed the roles of Setd2 in HCC and the underlying mechanisms in regulating cholesterol homeostasis and c-Jun/AP-1 signaling.

SETDB1, a histone H3K9 methyltransferase, has been reported to be upregulated in a variety of tumors and promotes cancer development. However, the exact pathogenesis of SETDB1 in human colorectal cancer (CRC) is hitherto unknown. Here, we showed that SETDB1 expression was highly amplified in CRC. Functionally, SETDB1 downregulation in SW480 and HCT116 cells reduced cell proliferation, migration, invasion, and increased CRC cells apoptosis. In contrast, SETDB1 overexpression promoted CRC cells proliferation, migration, and invasion. High expression of SETDB1 was associated with a more aggressive phenotype in vitro. Flow cytometry showed that cell cycle was arrested in G1 phase after SETDB1 silencing. Furthermore, depletion of SETDB1 in vivo suppressed CRC cells proliferation. Mechanistically, p21 was identified as the target of SETDB1. After transfected with siSETDB1, expression of p21 was distinctly increased. In contrast, expression of p21 was significantly decreased after overexpression SETDB1. We also showed that SETDB1 could be involved in the regulation of epithelial–mesenchymal transition (EMT) in HCT116 cells. Moreover, we confirmed that SETDB1 could regulate the activity of p21 promoter by dual-luciferase repoter assay, and proved that SETDB1 could bind to the promoter of p21 and regulate its H3K9me3 enrichment level by ChIP-PCR experiment. Finally, we verified that silencing of SETDB1 inhibited CRC tumorigenesis in vivo. In conclusion, our results indicate that SETDB1 is a major driver of CRC development and might provide a new therapeutic target for the clinical treatment of CRC.

Hippo pathway is involved in tumorigenesis, and its regulation in cytosol has been extensively studied, but its regulatory mechanisms in the nuclear are not clear. In the current study, using a FBS-inducing model following serum starvation, we identified KDM3A, a demethylase of histone H3K9me1/2, as a positive regulator for hippo target genes. KDM3A promotes gene expression through two mechanisms, one is to upregulate YAP1 expression, and the other is to facilitate H3K27ac on the enhancers of hippo target genes. H3K27ac upregulation is more relevant with gene activation, but not H3K4me3; and KDM3A depletion caused H3K9me2 upregulation mainly on TEAD1-binding enhancers rather than gene bodies, further resulting in H3K27ac decrease, less TEAD1 binding on enhancers and impaired transcription. Moreover, KDM3A is associated with p300 and required for p300 recruitment to enhancers. KDM3A deficiency delayed cancer cell growth and migration, which was rescued by YAP1 expression. KDM3A expression is correlated with YAP1 and hippo target genes in colorectal cancer patient tissues, and may serve as a potential prognosis mark. Taken together, our study reveals novel mechanisms for hippo signaling and enhancer activation, which is critical for tumorigenesis of colorectal cancer.

SPOP is one of the important subunits for CUL3/SPOP/RBX1 complex tightly connected with tumorigenesis. However, its exact roles in different cancers remain debatable. Here, we identify CYCLIN E1, as a novel substrate for SPOP. SPOP directly interacts with CYCLIN E1 and specific regulates its stability in prostate cancer cell lines. SPOP/CUL3/RBX1 complex regulates CYCLIN E1 stability through poly-ubiquitination. CDK2 competes with SPOP for CYCLIN E1 interaction, suggesting that SPOP probably regulates the stability of CDK2-free CYCLIN E1. CYCLIN E1 expression rescued proliferation, migration, and tumor formation of prostate cancer cell suppressed by SPOP. Furthermore, we found SPOP selectively regulates the substrates’ stability and signaling pathways in prostate cancer and CCRC cell lines, suggesting that complicated mechanisms exist for SPOP to regulate substrate specificity. Altogether, we have revealed a novel mechanism for SPOP in suppressing prostate cancer and provided evidence to show SPOP has dual functions in prostate cancer and CCRC.

ERAD is an important process of protein quality control that eliminates misfolded or unassembled proteins from ER. Before undergoing proteasome degradation, the misfolded proteins are dislocated from ER membrane into cytosol, which requires the AAA ATPase p97/VCP and its cofactor, the NPL4-UFD1 dimer. Here, we performed a CRISPR-based screen and identify many candidates for ERAD regulation. We further confirmed four proteins, FBOX2, TRIM6, UFL1 and WDR20, are novel regulators for ERAD. Then the molecular mechanism for WDR20 in ERAD is further characterized. Depletion of WDR20 inhibits the degradation of TCRα, a typical ERAD substrate, while WDR20 overexpression reduces TCRα protein level. WDR20 associates with TCRα and central regulators of the ERAD system, p97, GP78 and HRD1. A portion of WDR20 localizes to the ER-containing microsomal membrane. WDR20 expression increases TCRα ubiquitination, and HRD1 E3 ligase is essential for the process. WDR20 seems to serve as an adaptor protein to mediate the interaction between p97 and TCRα. Our study provides novel candidates and reveals an unexpected role of WDR20 in ERAD regulation.

Enhancers are cis-acting elements with many sites bound by transcription factors and activate transcription over long distance. Histone modifications are critical for enhancer activity and utilized as hallmarks for the identification of putative enhancers. Monomethylation of histone H3 lysine 4 (H3K4me1) is the mark for enhancer priming; acetylation of histone H3 lysine 27 (H3K27ac) for active enhancers and trimethylation of histone H3 lysine 27 (H3K27me3) for silent enhancers. Recent studies from multiple groups have provided evidence that enhancer reprogramming, especially gain of enhancer activity, is closely related to tumorigenesis and cancer development. In this review, we will summarize the recent discoveries about enhancer regulation and the mechanisms of enhancer reprogramming in tumorigenesis, and discuss the potential application of enhancer manipulation in precision medicine.

Although the exact etiology of inflammatory bowel disease (IBD) remains unclear, exaggerated immune response in genetically predisposed individuals has been reported. Th1 and Th17 cells mediate IBD development. Macrophages produce IL-12 and IL-23 that share p40 subunit encoded by IL12B gene as heteromer partner to drive Th1 and Th17 differentiation. The available animal and human data strongly support the pathogenic role of IL-12/IL-23 in IBD development and suggest that blocking p40 might be the potential strategy for IBD treatment. Furthermore, aberrant alteration of some cytokines expression via epigenetic mechanisms is involved in pathogenesis of IBD. In this study, we analyzed core promoter region of IL12B gene and investigated whether IL12B expression could be regulated through targeted epigenetic modification with gene editing technology. Transcription activator-like effectors (TALEs) are widely used in the field of genome editing and can specifically target DNA sequence in the host genome. We synthesized the TALE DNA-binding domains that target the promoter of human IL12B gene and fused it with the functional catalytic domains of epigenetic enzymes. Transient expression of these engineered enzymes demonstrated that the TALE-DNMT3A targeted the selected IL12B promoter region, induced loci-specific DNA methylation, and down-regulated IL-12B expression in various human cell lines. Collectively, our data suggested that epigenetic editing of IL12B through methylating DNA on its promoter might be developed as a potential therapeutic strategy for IBD treatment.

Histone positioning and modifications on viral genomes are important factors regulating virus replication. To investigate the dynamics of modified histones on the viral genome and their potential roles in antiviral response, we studied the dynamic changes of histone modifications across the HSV-1 genome in THP-1 cells. Histone modifications were detected on the HSV-1 genome soon after infection, including H3K9me3, H3K27me3, H3K4me3 and H3K27ac. These modifications emerged on the viral genome soon after infection and changed rapidly along with virus life cycle progression. The transcription repression marks, H3K9me3 and H3K27me3, decreased on the viral genome during the infection process; the transcription activation mark H3K27ac increased. Treatment with C646, an inhibitor of H3K27ac transferase p300, significantly repressed virus replication and viral gene expression. Our study reveals the relationship between histone modifications and viral gene expression and provides potential novel strategies for antiviral treatment.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract. In current dogma, pathogenesis of IBD is attributed to the dysregulated mucosal immune response to gut flora in genetically susceptible individuals, but the genetics evidence from GWAS studies so far is insufficient to explain the observed heritability in IBD. For this discordance, epigenetics has emerged to be one of the important causes. Recent studies have reported that histone acetylation is correlated with the development of IBD, whereas its role and underlying molecular mechanism in the disease still remain elusive. Here, we established a dextran sulfate sodium (DSS)-induced chronic colitis model and performed RNA-sequencing (RNA-seq) and Chromatin Immunoprecipitation followed by NGS sequencing (ChIP-seq) for H3K27ac in the mice colon tissues to investigate whether H3K27ac is involved in the development of intestinal inflammation. We found that the global H3K27ac level and distribution in colon tissue had no significant difference after DSS treatment, while H3K27ac signals were significantly enriched in the typical-enhancers of the DSS group compared with the control. By combining with RNA-seq data (fold change >2), we identified 56 candidate genes as potential target genes for H3K27ac change upon DSS treatment. We further predicted transcription factors (TFs) involved in DSS-induced colitis according to the enhancers with increased H3K27ac. H3K27ac increase in special typical-enhancers in the DSS group is possibly related to the development of intestinal inflammation by up-regulating adjacent gene expression and shifting TF networks, which will provide new insight into the pathogenesis and therapy of IBD.

Trimethylation of histone H3K36 is a chromatin mark associated with active gene expression, which has been implicated in coupling transcription with mRNA splicing and DNA damage response. SETD2 is a major H3K36 trimethyltransferase, which has been implicated as a tumor suppressor in mammals. Here, we report the regulation of SETD2 protein stability by the proteasome system, and the identification of SPOP, a key subunit of the CUL3 ubiquitin E3 ligase complex, as a SETD2-interacting protein. We demonstrate that SPOP is critically involved in SETD2 stability control and that the SPOP/CUL3 complex is responsible for SETD2 polyubiquitination both in vivo and in vitro. ChIP-Seq analysis and biochemical experiments demonstrate that modulation of SPOP expression confers differential H3K36me3 on SETD2 target genes, and induce H3K36me3-coupled alternative splicing events. Together, these findings establish a functional connection between oncogenic SPOP and tumor suppressive SETD2 in the dynamic regulation of gene expression on chromatin.

Prof. Mei YE works in the Department of Gastroenterology, Zhongnan Hospital, Wuhan University, and Hubei Clinical Center and Key Lab of Intestinal and Colorectal Diseases, as shown above. Her affiliation was incorrectly indicated in the article.

Adipic acid(AA) has a wide range of industrial applications and is enormously demanded. However, there are many disadvantages such as high energy consumption, environmental pollution in the current industrial process. Development of environment-friendly and effective synthetic methods is in urgent need. This review mainly introduces the latest research progresses of green synthetic methods of adipic acid by direct oxidation of cyclohexane using clean oxidants such as O2 (air), H2O2 and O3.

Upon virus infection, retinoic acid–inducible gene I–like receptors in host cells recognize viral RNA and activate type I IFN expression. Previously, we identified WD repeat domain (WDR) 5 as one positive regulator for pathway activation. In this study, we report that WDR82, a homolog protein of WDR5, acts opposite to WDR5 and inhibits the activation of the retinoic acid–inducible gene I signaling pathway. WDR82 overexpression inhibits virus-triggered pathway activation, whereas its knockdown enhances induced IFN-β expression. WDR82 is localized on the mitochondria, and its first N-terminal WD40 domain is critical for localization. WDR82 interacts with TNFR-associated factor (TRAF) 3, and its overexpression promotes K48-linked, but not K63-linked, polyubiquitination on TRAF3. Furthermore, WDR82 knockdown inhibits viral replication in the cell, whereas its overexpression has the opposite effect. Interestingly, WDR82 regulates Sendai virus–induced IFNB1 expression in a cell type–specific manner. Taken together, our findings demonstrate that WDR82 is a negative regulator of virus-triggered type I IFNs pathway through mediating TRAF3 polyubiquitination status and stability on mitochondria.

MicroRNAs (miRNAs) exhibit aberrant expression in the initiation and progression of a variety of human cancers, including colorectal cancer (CRC). However, the exact mechanisms are not well defined. miRNA expression profiles were characterized by microarrays in CRC samples, and miRNA 18b (miR-18b) was increased significantly in tumor tissues. The expression of miR-18b was confirmed in the CRC cell lines SW480 and HCT116 and 44 clinical specimens by quantitative real-time PCR (qRT-PCR). Multiple linear regression analysis showed a strong correlation of miR-18b expression with lymph node and distant metastasis. Overexpression of miR-18b promoted cell proliferation by facilitating cell cycle progression, and knockdown of miR-18b significantly suppressed migration in CRC cells. CDKN2B was identified as a target of miR-18b by high-throughput RNA sequencing and bioinformatics. After transfection with a miR-18b mimic, expression of CDKN2B was reduced significantly in CRC cells, and the effect was restored when a miR-18b inhibitor was transfected. A luciferase assay indicated miR-18b directly binds to the 3′ untranslated region (UTR) of CDKN2B. Expression of CDKN2B was downregulated in patient cancer tissues and negatively correlated with miR-18b. In a model of ectopic expression of miR-18b and CDKN2B, CDKN2B overexpression antagonized the effects of miR-18b in vitro and in vivo. The data show that miR-18b is involved in CRC carcinogenesis through targeting CDKN2B.

Lian-Yun Li,1,2 Ruo-Lan Ma,3 Liqin Du,4 An-Shi Wu5 1Department of Anesthesiology, Dongfang Hospital of Beijing University of Chinese Medicine, 2Department of Pain, Beijing Electric Power Hospital, 3Department of Anesthesiology, Beijing Stomatological Hospital, Capital Medical University, Beijing, People’s Republic of China; 4Department of Chemistry & Biochemistry, Texas State University, San Marcos, TX, USA; 5Department of Anesthesiology, Beijing Chao-Yang Hospital, Capital Medical University, Beijing, People’s Republic of China Objectives: Ozonated autohemotherapy (O3-AHT) has been used to effectively treat gout, but the underlying therapeutic mechanisms remain unknown. In this study, as an initial effort to understand the therapeutic mechanisms of O3-AHT, we aim to examine the effect of O3-AHT on serum inflammatory cytokine levels in gouty patients. Patients and methods: Three groups of patients and healthy subjects were recruited, including the gouty (n=10), hyperuricemia (n=10), and healthy control (n=11) groups. Cytometric bead array was applied to examine 12 cytokines before (T0), during (T1), and after (T2) therapies. Results: Three cytokines, IL-8, IL-12, and MCP-1, were detectable in all participants. Before O3-AHT, the average serum levels of IL-8 and MCP-1 were higher in the gout group than in the hyperuricemia and healthy control groups, confirming the inflammation status in gouty patients. After the 5th course of O3-AHT (T1), IL-8 level was significantly increased compared to that at T0. IL-12 level was also raised at T1, although the difference did not reach statistical significance. After completing the therapy, both IL-8 and IL-12 levels decreased to levels lower than those at T0. MCP-1 level remained essentially unchanged during and after treatment. Conclusion: Our results indicate that O3-AHT induces a significant change in serum cytokine levels, suggesting that modulating the inflammatory process is one of the therapeutic mechanisms underlying O3-AHT. In addition, the sensitive response of serum IL-8 and IL-12 levels to O3-AHT suggests that these cytokines may be developed as biomarkers to evaluate the therapeutic effect of O3-AHT in gouty patients. Keywords: gout, ozonated autohemotherapy, inflammation, cytokine

Aim: Epigenetic marks are critical regulators of chromatin and gene activity. Their roles in normal physiology and disease states, including cancer development, still remain elusive. Herein, the epigenomic change of H3K9me3, as well as its potential impacts on gene activity and genome stability, was investigated in an in vitro breast cancer transformation model. Methods: The global H3K9me3 level was studied with western blotting. The distribution of H3K9me3 on chromatin and gene expression was studied with ChIP-Seq and RNA-Seq, respectively. Results: The global H3K9me3 level decreases during transformation and its distribution on chromatin is reprogrammed. By combining with TCGA data, we identified 67 candidate oncogenes, among which five genes are totally novel. Our analysis further links H3K9me3 with transposon activity, and suggests H3K9me3 reduction increases the cell’s sensitivity to DNA damage reagents. Conclusion: H3K9me3 reduction is possibly related with breast cancer transformation by regulating gene expression and chromatin stability during transformation.

Epigenetic factors and related small molecules have emerged to be strongly involved in autophagy process. Here we report that 2-PCPA and GSK-LSD1, two inhibitors of histone H3K4 demethylase KDM1A/LSD1, induce autophagy in multiple mammalian cell lines. The two small molecules induce accumulation of LC3II, formation of autophagosome and autolysosome, and SQSTM1/p62 degradation. 2-PCPA treatment inhibits cell proliferation through cell cycle arrest but does not inducing cell death. Exogenous expression of KDM1A/LSD1 impaired the autophagic phenotypes triggered by 2-PCPA. The autophagy induced by 2-PCPA requires LC3-II processing machinery. But depletion of BECN1 and ULK1 with siRNA did not affect the LC3-II accumulation triggered by 2-PCPA. 2-PCPA treatment induces the change of global gene expression program, including a series of autophagy-related genes, such as SQSTM1/p62. Taken together, our data indicate that KDM1A/LSD1 inhibitors induce autophagy through affecting the expression of autophagy-related genes and in a BECN1-independent manner.

Odontoblasts derive from neural crest–derived odontogenic mesenchymal cells, and they are an important barrier of defense for the host. Survival and immunity of odontoblasts play important roles in protecting the dentin-pulp structure. Autophagy can eliminate damaged organelles and recycle cellular components to facilitate cellular homeostasis. Autophagy can be activated with external stressors, such as starvation, hypoxia, and infection. In this study, the role of autophagy in inflamed odontoblasts was explored, and its possible mechanism was investigated. Cell viability was not affected by mild lipopolysaccharide (LPS) stimulation, and autophagy was activated during this process. Immunofluorescence of light chain 3 confirmed that autophagy was induced with LPS treatment. Early-stage autophagy inhibition resulted in down-regulated cell viability, contrary to the up-regulated cell viability at late-stage autophagy inhibition. Western blot suggested that p-Akt and survivin were not activated in the early stage, and they gradually increased and peaked in the late stage. Meanwhile, autophagy was down-regulated through the Akt/mTOR/survivin pathway in the late stage. Thus, autophagy has a dual role in inflamed odontoblasts, which indicates its importance in maintaining the microenvironment homeostasis of odontoblasts. Autophagy was induced as a survival mechanism in the early stage, and it decreased through the Akt/mTOR/survivin signaling pathway in the late stage.

Gout is a common form of arthritis; however, there are currently no effective therapies available. Ozonated autohemotherapy (O3-AHT) is a controversial, but successful method of treatment for a number of diseases. The present study is the first pilot study investigating the application of O3-AHT in patients with hyperuricemia and gout. In total, 10 patients diagnosed with gout were recruited and subjected to O3-AHT. Self-reported pain visual analog scale (VAS) scores and creatinine clearance values were evaluated prior to (T0), during (after the fifth session of O3-AHT treatment; 1-4 weeks; T1) and following the treatment course (5-28 weeks; T2). At T1, the creatinine clearance rate of the patients significantly increased from 105.14±35.33 (T0) to 121.45±44.52 ml/min (t=2.165, P=0.062), while the pain VAS score decreased from 5.35±2.78 (T0) to 3.30±2.21 (t=2.004, P=0.076). However, at T2, the creatinine clearance rate decreased slightly to 111.15±36.52 ml/min, and no statistically significant difference was observed from the value at T0 (t=1.723, P=0.123). The pain VAS score further decreased to 2.30±2.66 (t=2.628, P=0.027). In conclusion, O3-AHT decreased the creatinine clearance rate and the pain VAS scores of patients with hyperuricemia and gout; thus, may be a potential effective therapeutic approach.

The Polycomb group (PcG) genes repress gene expression mainly through chromatin modifications and regulation of chromatin structure. At present, at least four protein complexes of PcG proteins are identified, including Polycomb repressive complex 1 (PRC1), Polycomb repressive complex 2 (PRC2), PHO-repressive complex (PhoRC) and Polycomb repressive deubiquitinase (PR-DUB). In this review, the recent discoveries of the composition of the above complexes, as well as their roles in regulating histone modifications and gene silencing are discussed. We mainly focus on the composition of PRC1 and PRC2 complex and recruitment of PcG to target genes and mechanisms of PRC1 and PRC2-mediated gene silencing. Although much progress has been made in understanding gene silencing mediated by PcG proteins, we also discuss several important questions that still remained unanswered, such as the inheritance of histone modifications during cell division.

Upon virus infection, host cells use retinoic-acid–inducible geneI I (RIG-I)–like receptors to recognize viral RNA and activate type I IFN expression. To investigate the role of protein methylation in the antiviral signaling pathway, we screened all the SET domain–containing proteins and identified TTLL12 as a negative regulator of RIG-I signaling. TTLL12 contains SET and TTL domains, which are predicted to have lysine methyltransferase and tubulin tyrosine ligase activities, respectively. Exogenous expression of TTLL12 represses IFN-β expression induced by Sendai virus. TTLL12 deficiency by RNA interference and CRISPR-gRNA techniques increases the induced IFN-β expression and inhibits virus replication in the cell. The global gene expression profiling indicated that TTLL12 specifically inhibits the expression of the downstream genes of innate immunity pathways. Cell fractionation and fluorescent staining indicated that TTLL12 is localized in the cytosol. The mutagenesis study suggested that TTLL12’s ability to repress the RIG-I pathway is probably not dependent on protein modifications. Instead, TTLL12 directly interacts with virus-induced signaling adaptor (VISA), TBK1, and IKKε, and inhibits the interactions of VISA with other signaling molecules. Taken together, our findings demonstrate TTLL12 as a negative regulator of RNA-virus–induced type I IFN expression by inhibiting the interaction of VISA with other proteins.

Background and aim Emerging evidences indicate that small nucleolar RNAs (snoRNAs) are important regulatory molecules involved in various pathophysiological processes including inflammation and cancer. In the current study, we investigate whether snoRNAs dysregulate in colorectal cancer (CRC) and intestinal inflammation and contribute the pathogenesis of CRC. Methods We analyzed the snoRNAs expression profile in CRC patients by GeneChipR Array and validated candidate snoRNAs expression in 44 CRC tissues, as well as in 28 ulcerative colitis (UC) and 28 healthy controls using reverse transcription quantitative polymerase chain reaction. Furthermore, we analyzed the correlation between snoRNAs expression and clinical characteristics of CRC and evaluated the diagnosis and differentiation efficiencies of the snoRNAs in CRC and UC. Results The expression of snoRA15, snoRA41 displayed increased, whereas snoRD33 was down-regulated in CRC compared with matched non-cancerous tissues. When compared to healthy control, the three snoRNAs are all upregulated in lesion tissue of UC and CRC, which showed an increasingly trend from healthy control to UC and CRC. Conclusions The identified three snoRNAs might contribute the carcinogenesis of colorectal cancer and involve in the progress from chronic intestinal inflammation to malignant tumor.

Background Epigenetic regulation has emerged to be the critical steps for tumorigenesis and metastasis. Multiple histone methyltransferase and demethylase have been implicated as tumor suppressors or oncogenes recently. But the key epigenomic events in cancer cell transformation still remain poorly understood. Methods A breast cancer transformation model was established via stably expressing three oncogenes in primary breast epithelial cells. Chromatin immunoprecipitation followed by the next-generation sequencing of histone methylations was performed to determine epigenetic events during transformation. Western blot, quantitative RT-PCR, and immunostaining were used to determine gene expression in cells and tissues. Results Histones H3K9me2 and me3, two repressive marks of transcription, decrease in in vitro breast cancer cell model and in vivo clinical tissues. A survey of enzymes related with H3K9 methylation indicated that KDM3A/JMJD1A, a demethylase for H3K9me1 and me2, gradually increases during cancer transformation and is elevated in patient tissues. KDM3A/JMJD1A deficiency impairs the growth of tumors in nude mice and transformed cell lines. Genome-wide ChIP-seq analysis reveals that the boundaries of decreased H3K9me2 large organized chromatin K9 modifications (LOCKs) are enriched with cancer-related genes, such as MYC and PAX3. Further studies show that KDM3A/JMJD1A directly binds to these oncogenes and regulates their transcription by removing H3K9me2 mark. Conclusions Our study demonstrates reduction of histones H3K9 me2 and me3, and elevation of KDM3A/JMJD1A as important events for breast cancer, and illustrates the dynamic epigenomic mechanisms during breast cancer transformation. Electronic supplementary material The online version of this article (doi:10.1186/s13148-016-0201-x) contains supplementary material, which is available to authorized users.

Endoplasmic reticulum (ER)-associated degradation (ERAD) of unassembled T-cell receptor alpha-chain (TCRalpha) is reliant on the presence of two basic residues in the transmembrane (TM) segment of TCRalpha. The precise role of these residues in ER quality control is unclear. Here, we show that a TCRalpha mutant lacking these intramembrane charged residues has a tendency to form homooligomers through an interchain disulfide bond that involves a specific pair of cysteine residues. Covalent oligomerization of TCRalpha appears to stabilize it at the ER membrane. The presence of a single lysine residue at specific positions within the TCRalpha TM domain abolishes its oligomerization and causes its rapid degradation. Conversely, when TCRalpha oligomerization is induced by a bivalent compound, the degradation of TCRalpha is inhibited. Together, these results suggest that the intramembrane charged residues in TCRalpha do not function as a signal for substrate recognition in ERAD. Instead, their primary role is to reduce TCRalpha oligomerization, maintaining it in a retrotranslocation-competent state. Our results also suggest that the ERAD machinery is inefficient when coping with oligomerized substrates, indicating a requirement for chaperone-mediated protein disassembly in the ER lumen prior to retrotranslocation.

Unlike other members of the polycomb group protein family, EZH1 has been shown to positively associate with active transcription on a genome-wide scale. However, the underlying mechanism for this behavior still remains elusive. Here, we report that EZH1 physically interacts with UXT, a small chaperon-like transcription co-activator. UXT specifically interacts with EZH1 and SUZ12, but not EED. Similar to upon knockdown of UXT, knockdown of EZH1 or SUZ12 through RNA interference in the cell impairs the transcriptional activation of nuclear factor (NF)-κB target genes induced by TNFα. EZH1 deficiency also increases TNFα-induced cell death. Interestingly, chromatin immunoprecipitation and the following next-generation sequencing analysis show that H3K27 mono-, di- and tri-methylation on NF-κB target genes are not affected in EZH1- or UXT-deficient cells. EZH1 also does not affect the translocation of the p65 subunit of NF-κB (also known as RELA) from the cytosol to the nucleus. Instead, EZH1 and SUZ12 regulate the recruitment of p65 and RNA Pol II to target genes. Taken together, our study shows that EZH1 and SUZ12 act as positive regulators for NF-κB signaling and demonstrates that EZH1, SUZ12 and UXT work synergistically to regulate pathway activation in the nucleus.

Electron-rich dienes have revolutionized the synthesis of complex compounds since the discovery of the legendary Diels–Alder cycloaddition reaction. This highly efficient bond-forming process has served as a fundamental strategy to assemble many structurally formidable molecules. Amino silyloxy butadienes are arguably the most reactive diene species that are isolable and bottleable. Since the pioneering discovery by Rawal, 1-amino-3-silyloxybutadienes have been found to undergo cycloaddition reactions with unparalleled mildness, leading to significant advances in both asymmetric catalysis and total synthesis of biologically active natural products. In sharp contrast, this class of highly electron-rich conjugated olefins has not been studied in non-cycloaddition reactions. Here we report a simple synthesis of tetrasubstituted 1-silyloxy-3-aminobutadienes, a complementarily substituted Rawal's diene. This family of molecules is found to undergo a series of intriguing chemical transformations orthogonal to cycloaddition reactions. Structurally diverse polysubstituted ring architectures are established in one step from these dienes., Electron-rich dienes have been shown to have multiple uses in synthesis by means of cycloaddition reactions. Here, the authors report a series of non-cycloaddition-based reactions of these compounds, giving access to 4-, 5-, 6- and 7-membered ring systems.

Toll-like receptor 3 (TLR3) recognizes dsRNA generated during viral infection and activation of TLR3 results in induction of type I interferons (IFNs) and cellular anti-viral response. TLR3 is associated with a TIR domain-containing adapter protein TRIF, which activates distinct downstream pathways leading to activation of NF-kappaB and ISRE sites in the promoters of type I IFNs. We show here that A20, a NF-kappaB-inducible zinc finger protein that has been demonstrated to be an inhibitor of TNF-induced NF-kappaB activation and a physiological suppressor of inflammatory response, potently inhibited TLR3- and Sendai virus-mediated activation of ISRE and NF-kappaB and IFN-beta promoter in reporter gene assays. A20 also inhibited TRIF-, but not its downstream signaling components TBK1-, IKKbeta-, and IKKepsilon-mediated activation of ISRE and NF-kappaB and IFN-beta promoter. Moreover, A20 interacted with TRIF in co-immunoprecipitation experiments. Finally, expression of A20 could be induced at protein level by Sendai virus infection. These data suggest that A20 targets TRIF to inhibit TLR3-mediated induction of IFN-beta transcription and functions as a feedback negative regulator for TLR3 signaling and cellular anti-viral response.